The present invention relates to sample substrates, such as plates, slides and cells, for use in examining, indicating, analyzing or identifying fluorescent, phosphorescent or luminescent sample materials, e.g. tagged molecular biological specimens, and in particular relates to such sample holders whose optical structures are adapted for enhancing fluorescence detection and imaging. The format of the sample substrates of the present invention can be adapted to formats typically used in this field, such as for example the standard format of microtiter plates.
Fluorescence microscopy is often used in the fields of molecular biology, biochemistry and other life sciences for analyzing biological molecules, including nucleic acids (DNA, RNA) and proteins (enzymes, antigens, etc.) that have been tagged or labeled with fluorescent probes. One such use is DNA diagnostics, such as for gene detection, in which a DNA sample is deposited on and bound to a glass substrate. The bound DNA on the substrate can then be imaged by fluorescence. The fluorescence of a sample was originally assessed by visual inspection through a conventional microscope, but this manual method has proved time-consuming and costly. Many different high-speed automated fluorescence imaging systems are now available.
An important figure of merit for fluorescence detection and measurement instruments is sensitivity, which is primarily determined by the signal-to-noise ratio (SNR) of the optical imaging system of the instrument. A well-designed imaging system has a signal-to-noise ratio that is limited by its light collection ability and not by internal noise sources. The theoretical SNR of such a system is expressed in terms of the number of photoelectrons at the cathode when using a photomultiplier tube (PMT), which in turn essentially depends upon the number of photons that reach the detector from the area of interest on the sample substrate, the quantum efficiency of the detector, and the number of dark electrons generated by the detector.
One obvious approach to increasing SNR, and thereby improving sensitivity, is to reduce background noise. Sources of background noise include specular or diffuse reflection of the fluorescence-stimulating laser light from the sample, autofluorescence of the substrate holding the sample, autofluorescence from the optics in the light path of the optical imaging system, stray light, and dark current of the detector. Stray light reaching the detector can be significantly reduced by proper size and placement of apertures in the imaging system. Both stray light and much of the reflected laser light can be rejected, while passing the fluorescent light, by using dichroic and other spectral filters and beamsplitters in the system. Autofluorescence of the optical elements can be reduced by avoiding use of lens cements in the light path, using glass instead of polymeric lenses, or using curved mirrors instead of lenses wherever possible.
Autofluorescence of the substrate can be reduced by using low fluorescence materials, such as an ultrathin or opaque glass substrate. For example, in U.S. Pat. No. 5,095,213 Strongin discloses a plastic slide that is rendered opaque and substantially nonfluorescent with a quantity of black carbon powder in the plastic. Another way of handling autofluorescence is to use a pulsed or modulated excitation and to take advantage of the differences in emission decay rates between background fluorescence and specimen fluorescence, as disclosed in U.S. Pat. No. 4,877,965 to Dandliker et al. and U.S. Pat. No. 5,091,653 to Creager et al.
In U.S. Pat. No. 5,552,272, Bogart discloses an assay system and method for detecting the presence or amount of an analyte of interest. It includes a test substrate with an optically active surface that enhances the color contrast, i.e. differences in the observed wavelength (or combination of wavelengths) of light from the surface, between the presence and absence of the analyte in a sample applied onto the test substrate. In particular, the substrate may comprise a reflective solid optical base, such as a silicon wafer or metallic (e.g., aluminum) base, with an optical thin film coating thereon. The coating may comprise several layers, including for example an attachment layer on the upper surface of the base, and a receptive layer on the upper surface of the attachment layer containing a specific binding partner for the analyte of interest. The total coating thickness is selected to cause incident light to undergo thin film interference upon reflection, such that a specific color is produced. Specifically, the coating material(s) should have an overall thickness of a quarterwave of the unwanted color to be attenuated so that destructive interference of that color will occur. The substrate therefore has a particular background color, which can then be used as a comparative reference against a different observed color when an analyte of interest is present. Both qualitative visual inspection and quantitative instrumented measurement are suggested. Polarization contrast by means of an ellipsometer is also suggested.
One example of the use to which the Bogart invention has been put by Biostar, Inc. of Boulder, Colo., the assignee of the aforementioned patent, is an optical immunoassay (OIA) diagnostic screening test for the rapid detection (in under 30 minutes) of the presence of specific antigens of infectious pathogens in a sample taken from a patient. Commercial products include test kits for group A and group B streptococci and for chlamydia trachomatis. These particular assays are given as examples in the Bogart patent, are described in package inserts for the corresponding Biostar products and are also described in a number of published articles in medical journals. Briefly, they all rely on direct visual detection of a change in the color of light reflection off of the test substrate due to a physical change in the optical thickness of a molecular thin film coating on the substrate surface which results from binding reactions between an immobilized antibody on the test surface and a specific antigen that may be present in a drop of sample liquid applied to the test surface. The original bare test surface has a thin film thickness that results in a predominant visual background gold color when white light is reflected off of the surface. The antigen-antibody binding reaction that occurs when the specific antigen of interest is present in the applied sample results in an increase in the thin film thickness that causes a corresponding change in the color of the test surface from gold to purple. If on the other hand, the antigen is not present in the sample, no binding takes place, the original thin film thickness remains unchanged and the test surface retains its original gold color, indicating a negative result. This diagnostic assay tool is very sensitive and easily interpreted.
Bogart further discloses, in another embodiment of his invention (FIG. 17 of the aforementioned patent), the use of these substrates for enhanced fluorescence detection. After the analyte of interest has been bound to the surface by reaction with the specific binding partner in the receptive layer of the substrate coating, fluorescent label molecules may be attached to the analyte. In particular, the fluorescent molecules may be attached to any suitably selective and specific receptive material or reagent, such as a secondary antibody, and applied to the surface. The fluorescent labels are thus bound to the analyte of interest on the surface, if present, and immobilized to the surface through the analyte bridge. Directing light of an excitation wavelength onto the surface stimulates fluorescence of any of the label bound to the surface, thereby revealing the presence of the analyte of interest. Because the maximum fluorescence wavelength may not be shifted far enough from the excitation wavelength to be distinguished, the reflective substrate may have an antireflection layer whose thickness is selected to suppress reflection of the excitation wavelength, thereby reducing the background noise reaching the detector. Bogart states that the fluorescent signal generation is not dependent on the film thickness. Though the fluorescence signal S might be increased by increasing the output power of the laser, reflected laser noise will also increase, with possibly little improvement in the resulting SNR.
In U.S. Pat. No. 6,008,892 Kain et al disclose a sample substrate which is reflective for the excitation wavelength. This substrate has a transparent coating layer thereon with controlled thickness that has been selected to ensure that a molecular sample placed on top of the coating layer is located at an antinode for the excitation light. In particular, the substrate includes a rigid base with a specularly reflective upper surface. The transparent coating on the upper surface of the base has a thickness selected such that for a particular excitation wavelength of light at normal incidence, the optical path from the top of the coating to the base reflecting surface is substantially an odd multiple (1, 3, 5, etc.) of one-quarter wavelength of the excitation light. The optical path length of the material is defined by the wavelength of light, the index of refraction of the material, and the angle of propagation through the material. In the reflective sample substrate the reflecting surface of the base is at a well defined depth slightly below the physical surface of the base by an amount equal to the sum of the skin (or penetration) depth of the reflective surface material and the optical depth of any surface oxidation on the base. By placing the sample on the coating layer at or near the antinode of the excitation light, maximum fluorescence excitation occurs. A reflective substrate also enhances fluorescence collection by nearly doubling the solid collection angle of a fluorescence imaging microscope system. Thus, the total fluorescence signal is increased, leading to a much improved signal-to-noise ratio. Also, because the coating layer is very thin, there is reduced fluorescence background noise from this material.
As Kain et al describe the base can be made completely of metal or may be composed of a rigid bottom layer with a top metal coating. The metal can be aluminum, silver, gold or rhodium. The transparent coating may be a single layer of dielectric material, such as silica, alumina or a fluoride material (such as MgF2). Alternatively, the transparent coating could be a multilayer coating with the top layer being a chemically reactive material for binding a specified biological sample constituent thereto.
Kains concept of applying a transparent quarterwave layer on a reflecting surface is limited to reflectors with rigid surfaces. Here light is not penetrating beneath the physical surface of the reflector or the penetration is limited to at most some nanometers beneath the surface (skin depth). Practically this represents a limitation to metallic surfaces such as metal substrates or metallic coatings.
As soon as dielectric layers significantly contribute to the reflection, it is difficult to define a penetration depth and the concept of adding a quarterwave layer fails. For example for aluminum mirrors very often additional dielectric layers are used to enhance the reflectivity of the metal which results in so called “pumped metallic mirrors”. In this case the dielectric layers are part of the mirror and the last dielectric layer forms the physical surface of the mirror. Light penetrates into such a mirror stack and interference effects together with the metallic reflectivity establish the optical characteristics. Adding an additional quarterwave layer or an odd multiple to such a system very often fails to produce an antinode at the location of fluorescent sample when placed on this layer. In addition in most cases the interference system is disturbed which might result in a drastic decrease of reflectivity.
However as long as the odd multiple quarterwave condition is fulfilled with respect to the metallic surface it is even possible to enhance fluorescence using the reflectivitiy contribution of the quarterwave layers. Chaton et al give one example in WO 02/48691 where this effect can be seen. Chaton et al describe the use of dielectric quarterwave stacks which have a mirror function on a silicon substrate in order to enhance the fluorescence. The reflectivity of silicium at 550 nm is at about 42%. This results in a square electromagnetic field amplitude (E2) on the metallic surface of about 10% of the square of the field amplitude of the free propagating wave E2(PW). Applying a single quaterwave layer of SiO2 on that rigid metallic surface (following one embodiment of the invention of Kain et al) results in a field amplitude which is 25% higher than E2(PW) therefore resulting in an enhancement factor of 1.25. Chaton et al use a quarterwave stack with a design wavelength of 550 nm in order to reinforce reflectivity. This means that a system of alternating high and low index dielectric layers is used to increase the reflectivity, where each individual layer has an optical thickness of one quarter of the design wavelength. As an example Chaton et al use a five layer system on silicon with SiO2 (94 nm layer thickness, 3 layers) and Si3N4 (69 nm layer thickness, 2 layers) as coating materials. This results in a reflectivity of slightly below 60%. Mirrors based on quarterwave stacks are known as Bragg mirrors. Typically such a Bragg mirror is based on a quarterwave stack where the outermost layer is a high index layer. However Chaton et al use as outermost quarterwave layer an SiO2 layer, which is the low index material. It is known that the outermost layer being a low index layer results in lower reflection values. Removing the outermost layer (low index layer) would even result in higher reflectivity. As the Chaton et al describe they use SiO2 as outermost layer in order to create a physical surface which is compatible with the linker chemistry. However there is one additional positive effect the authors did not mention: Our investigations showed that using a low index layer as outermost layer results in a field amplitude for 550 nm on the surface which is fortunately maximum, 220% more than E2(PW) resulting in an enhancement factor of 2.2. If the authors had used a high index layer as outermost layer the electromagnetic field amplitude would have been minimum.
With a coating design as used by Chaton et al the reflection band is centered at the design wavelength of 550 nm and is 200 nm broad. Within the reflection band the reflectivity is increased to slightly below 60%. Because Chaton et al did not take into account the antinode condition, they conclude that the quarterwave stack is effectively enhancing the fluorescence signal for wavelengths between 450 nm to 650 nm. This wavelength range comprises the wavelengths of the fluorescence materials typically used for fluorescence lables such as CY3 and CY5. Unfortunately only for the design wavelength the antinode condition is well fulfilled. For an excitation wavelength much different from 550 nm this condition is not fulfilled. For 450 nm the enhancement factor is as low as 0.2 and for 650 nm the enhancement factor decreases down to 1.2.
It is therefore still a problem and would be desirable to create a sample which provides optimum enhancement for more than one excitation wavelength well separated from each other. Especially for the excitation wavelengths around 532 nm-548 nm (Cy3) and around 633 nm (Cy5) it is still an open question how to realize such a sample substrate.
Typically there is an inherent difference of signal intensity for Cy3 and Cy5 related for example to differences in affinity of the linker chemistry, resulting in different signal intensities. Therefore the question of how to realize for two or more excitation wavelengths approximately the same degree signal intensities may be important. It is an interesting, but even more general aspect to consider the possibility to adjust the enhancement factors for two or more excitation wavelengths independently.
In addition as discussed, the prior art solutions involve in general interfaces to metallic layers or substrates. Chaton for example restricts the discussion to a silicon substrate. Examples for rigid reflecting surfaces in Kains disclosure always involve metallic surfaces. It is therefore still a problem and would be desirable to create a substrate sample providing enhanced fluorescence signal without use of a metallic interface as do Kain et al as well as Chaton et al.